Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [³H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate > ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydro-kainate > 6, 7-dinitroquinoxaline-2, 3-dione > 6-cyano-7-nitroquinoxaline-2, 3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.     

Assimilation of ethylenediamine as nitrogen source by the cyanobiont Nostoc ANTH

Nostoc ANTH metabolizes ethylenediamine (EDA) as sole nitrogen source but not as a carbon source. EDA is assimilated by the glutamine synthetase-glutamate synthase pathway. EDA represses heterocyst formation and nitrogenase activity but this is reversed by l-methionine-dl-sulphoximine.The authors are with the Department of Microbiology, Barkatullah University, Bhopal 462 026, India     

海南“三亚人”遗址1992年发掘报告

本文记述的是海南三亚发现的石器时代洞穴遗址,地质时代为晚更新世末或全新世初期。哺乳动物组合为东洋界动物群,石制品在类型上或加工方式上与两广地区相同时代的文化类型比较接近。人类牙齿属晚期智人。     

Functional relationship between ammonia and gangliosides in brain

The functional significance of ammonia production in brain under physiological or pathological conditions is not clearly known. NH₄ ⁺ stimulates Na⁺, K⁺ activated ATPase causing stabilization of neuronal membranes of which gangliosides are major structural components. Moreover ammonia is known to inhibit lysosomal enzymes which include enzymes degrading gangliosides. Gangliosides have been shown to stimulate neuritogenesis in neuronal cultures and prevent the damage of the neurons from glutamate toxicity particularly in areas of brain ischemia. Hyperammonemia without any behavioural changes was induced in experimental rats by intraperitoneal administration of either a single dose (0.8 mmol/100 g wt.) or by six hourly doses (0.6 mmol/100 g wt.) of ammonium acetate. An increase in the content of gangliosides along with a rise in the content of GD_1A and GD_1B without any change in -galactosidase and N-acetylhexosaminidase was observed in cerebral cortex, cerebellum, and brain stem, following the administration of single dose of ammonium acetate. Gangliosides, after extraction from the different brain regions, were estimated by the thiobarbituric acid method and expressed in terms of sialic acid. Individual gangliosides were separated and estimated by thin layer chromatography using resorcinol as the staining agent. These results suggest that ammonia production in the neuronal pathways in brain either as a result of repeated stimulation under physiological conditions or as a result of focal ischemia or injury, may likewise cause an increase in the content of gangliosides which may help in neuritic growth (physiological conditions facilitating synaptic plasticity) and may exert a protective effect on the neurons in the ischemic area against glutamate toxicity.Former Professor of Biochemistry, OMC, Hyderabad.     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.     

Effects of Ca(II) ions on Mn(II) dynamics in chick glia and rat astrocytes: Potential regulation of glutamine synthetase

Previous studies have demonstrated that in glia and astrocytes Mn(II) is distributed with ca. 30–40% in the cytoplasm, 60–70% in mitochondria. Ca(II) ions were observed to alter both the flux rates and distribution of Mn(II) ions in primary cultues of chick glia and rat astrocytes. External (influxing) Ca(II) ions had the greatest effect on Mn(II) uptake and efflux, compared to internal (effluxing) or internal-external equilibrated Ca(II) ions. External (influxing) Ca(II) ions inhibited the net rate and extent of Mn(II) uptake but enhanced Mn(II) efflux from mitochondria. These observations differ from Ca(II)–Mn(II) effects previously reported with brain (neuronal) mitochondria. Overall, increased cytoplasmic Ca(II) acts to block Mn(II) uptake and enhance Mn(II) release by mitochondria, which serve to increase the cytoplasmic concentration of free Mn(II). A hypothesis is presented involving external L-glutamate acting through membrane receptors to mobilize cell Ca(II), which in turn causes mitochondrial Mn(II) to be released. Because the concentration of free cytoplasmic Mn(II) is poised near the K_d for Mn(II) with glutamine synthetase, a slight increase in cytoplasmic Mn(II) will directly enhance the activity of glutamine synthetase, which catalyzes removal of neurotoxic glutamate and ammonia.     

Evaluation of free D-glutamate in processed foods

Monosodium glutamate (MSG) is added to many processed foods at significant levels for flavor enhancement. It is also naturally occurring at high levels in some foods. The enantiomeric composition of free glutamate in foods was examined and all foods analyzed were found to contain D -glutamate. The relative percent of D -glutamate in the food products studied depended on the origin of the glutamate. Foods to which MSG was added by the manufacturer had a high total level of MSG but a lower relative percentage of the D -enantiomer (usually less than 0.8%). In comparison, fermented foods tend to have high relative levels of D -glutamate but a lower total amount of the amino acid. The relative percent of D -glutamate in nonfermented foods containing no added MSG was also found to be low compared to fermented products. In some cases the percent D -glutamate could be related to the relative amounts of other food ingredients such as cheese. © 1994 Wiley-Liss, Inc.     

Observations on the influence of glutamine,asparagine and peptone on growth and t-PA production of Chinese hamster ovary (CHO) cells

When a transfected CHO cell, that produces tissue-type Plasminogen Activator, t-PA, was transferred from a medium based on 5% Fetal Calf Serum, FCS, to a medium based on 0.8% casein peptone with variable glutamine and asparagine content, it was observed, that the growth of the cells changed from anchorage dependant to suspension culture giving more reproducible cultivations. In the FCS culture t-PA was unstable, observed as a decline in t-PA concentration after 250 h. This decline in t-PA concentration was not observed in the serum free culture, although there was a decline in productivity after 200 h. This change in production profile may be attributed to either no proteolytic attack from serum or by scavenging of proteolytic activities produced by the cells from the peptone peptides. Increasing amounts of glutamine/asparagine gave higher production of t-PA in synchrony with an increasing production of ammonia/ammonium ions. Ammonia inhibition does not seem to be a key factor for this cell line as seen with many others.     

Displacement and Return Movement of Chloroplasts in the Marine Dinophyte Pyrocystis noctiluca. Experiments with Optical Tweezers

Infrared laser traps (optical tweezers) were used to study laser-induced organelle movements in the marine alga Pyrocystis noctiluca (Dinophyta). These cells are highly suitable for optical micromanipulation due to their large size and extensive vacuole. Experiments were done with plastids held by optical tweezers and moved from the nuclear area into the vacuole. The subsequent retraction movement was analysed for speed. The displaced organelles remained connected to their original position by a thin cytoplasmic strand, often less than 1 μm in diameter. When the organelles were released they rapidly returned at an initial rate of 81.7 ± 7.8 μm . s^?1 (overall displacement 50 μm, measured distance 20 μm, 25 °C ± 1 °C, number of cells 22), slowing down with progressive retraction of the connecting strand. The return movement was reduced to 4.2 ± 0.2 μ .s^?1 (n = 10) when the organelles were displaced and held for 1 min. Displacement to a longer distance increased the rate of return movement. A change from a high to a low environmental temperature significantly reduced movement from 94.5 ± 9.0 . s^?1 (30 °C ± 1 °C, n = 22) to 34.5 ± 2.7 μm .s^?1 (5°C ± 1 °C, n = 22). Nocodazole and N-ethylmaleimide (NEM), inhibitors of microtubules and acto-myosin, respectively, did not affect the retraction of the connecting strand, but at high concentrations of NEM it became increasingly difficult to move organelles away from the nuclear area. We suggest that the return movement of organelles within laser-induced artificial strands mainly depends on the viscoelastic properties of the tonoplast. The quantification of these properties by optical tweezers allows determination of reactions of plant cells to temperature changes.